The development of an effective anti-HIV vaccine will greatly depend on our ability to elicit both cellular and humoral anti-viral immune responses, in the periphery and at mucosal sites. The generation of potent neutralizing antibody responses against diverse primary HIV isolates has not yet been fully achieved. Several reasons may account for this lack of success, including the form of the immunogen and the immunization methodology. The overall aim of this Project 1 is to design and evaluate novel HIV envelope (Env)-based immunogens capable of eliciting potent neutralizing antibody responses against heterologous primary HIV isolates. Our efforts will focus on using two complementary approaches to expose multiple neutralization epitopes that are conserved among primary HIV isolates. In the first, we plan to increase the exposure of conserved cryptic epitopes participating in Env-CD4 and ---chemokine receptor-binding through the introduction of deletions of the variable (V)-regions and "bridging-sheet" structures in the Env surface glycoprotein. In the second, we seek to expose these epitopes through the use of Env glycoproteins complexed to novel rationally-designed CD4 miniproteins. Env antigens from both subtype B and C HIV-1 isolates will be studied. We will employ a stepwise systematic approach to characterize the resulting Env immunogens and the antibody responses generated to them. This will involve pre-screening of candidate immunogens for expression, CD4-binding, and binding to a panel of monoclonal antibodies of known specificities. Once a candidate has met well-defined structural criteria, it then will be advanced to rabbit immunogenicity studies. Both DNA prime-protein boost and adjuvanted protein-alone vaccine regimens will be employed. Lead candidate Env immunogens and regimens will be further advanced to vaccine/challenge studies in primates based on their relative abilities to induce neutralizing antibody responses against primary HIV-1 strains. Secondary criteria will include the induction of high titer and/or high avidity antigen-specific antibodies directed against conformational epitopes. Vaccine/challenge studies will be performed in rhesus macaques using both homologous and heterologous pathogenic SHIV challenges. These studies should yield important information for the design of next generation HIV vaccines for future clinical evaluations.